4710 ONCOLOGY LETTERS 17: 4710-4716, 2019

L‑Lactate dehydrogenase B may be a predictive marker for sensitivity to anti‑EGFR monoclonal antibodies in cell lines

AYUMU NAGAMINE1,2, TAKUYA ARAKI1,2, DAISUKE NAGANO1, MITSUE MIYAZAKI3 and KOUJIROU YAMAMOTO1,2

1Department of Clinical Pharmacology and Therapeutics, Gunma University Graduate School of Medicine; 2Department of Pharmacy, Gunma University Hospital; 3Division of Endocrinology Metabolism and Signal Research, Gunma University Initiative for Advanced Research and Institute for Molecular and Cellular Regulation, Maebashi, Gunma 371‑8511, Japan

Received October 15, 2018; Accepted January 31, 2019

DOI: 10.3892/ol.2019.10075

Abstract. Recently, derived from cancer cells have Introduction been widely investigated as biomarkers for predicting the efficacy of chemotherapy. In this study, to identify a sensitive Various factors such as genetic variations and changes in biomarker for the efficacy of anti‑epidermal growth factor mRNA expression patterns are known to influence drug effi- receptor monoclonal antibodies (anti‑EGFR mAbs), proteins cacy, and such factors have been studied extensively. However, derived from 6 colorectal cancer (CRC) cell lines with drug responses are affected by changes in the conformation, different sensitivities to cetuximab, an anti‑EGFR mAb, were localization, and expression of numerous proteins, which analyzed. Cytoplasmic and membrane proteins extracted from are regulated by mutations and mRNA expression levels. In each CRC cell line were digested using trypsin and analyzed 2014, Zhang et al reported that only 32% of the showed comprehensively using mass spectrometry. As a result, 148 and statistically significant positive mRNA‑ correlation 146 peaks from cytoplasmic proteins and 363 and 267 peaks in 86 CRC samples (1). Therefore, proteomics analysis‑the from membrane proteins were extracted as specific peaks for direct evaluation of the expression levels and modifications of cetuximab‑resistant and ‑sensitive CRC cell lines, respectively. proteins‑has been focused on recently as a powerful explora- By analyzing the proteins identified from the peptide peaks, tion method for identifying predictive biomarkers for the cytoplasmic L‑lactate dehydrogenase B (LDHB) was detected efficacy of chemotherapeutic drugs. as a marker of cetuximab sensitivity, and it was confirmed The expression levels of some serum proteins have been that LDHB expression was increased in cetuximab‑resistant reported to be useful indicators of sensitivity to chemotherapy CRC cell lines. Furthermore, LDHB expression levels were for cancer, and Li et al reported that variation in serum LDH significantly upregulated with the acquisition of resistance to level was useful as a predictive biomarker of efficacy of beva- cetuximab in cetuximab‑sensitive CRC cell lines. In conclu- cizumab in non‑small cell lung cancer (NSCLC) patients (2). sion, LDHB was identified as an important factor affecting Furthermore, proteomic studies analyzing several serum cetuximab sensitivity using comprehensive proteome analysis proteins using matrix‑assisted laser desorption/ionization for the first time. mass spectrometry (VeriStrat; Biodesix, Boulder, CO), clas- sified NSCLC patients treated with erlotinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, into two groups with good or poor prognosis (3‑10). However, serum proteins derived from tumor tissue and circulating in blood stably represent only a small fraction of total protein Correspondence to: Dr Takuya Araki, Department of Clinical derived from a tumor. Thus, to find more suitable predictive Pharmacology and Therapeutics, Gunma University Graduate School biomarkers for sensitivity to anticancer drugs, the analysis of Medicine, 3‑39‑22 Showa‑machi, Maebashi, Gunma 371‑8511, should include not only proteins released into blood but all Japan E‑mail: tkyaraki@gunma‑u.ac.jp proteins derived from a tumor. In 2015, Sun et al (11) reported that a high level of L‑lactate Key words: L‑lactate dehydrogenase B, anti‑epidermal growth dehydrogenase B (LDHB) expression in tumor tissue was factor receptor monoclonal antibodies, colorectal cancer, effect associated with poor overall survival in oral cancer patients predictor, proteome treated with paclitaxel, and Ferrer et al (12) also reported that 17 kDa membrane‑associated protein expression in tumor tissue could predict sensitivity to platinum‑based therapy, EGFR inhibitors, and the proteasome inhibitor bortezomib in NAGAMINE et al: LDHB AS A PREDICTIVE MARKER FOR SENSITIVITY TO ANTI-EGFR MONOCLONAL ANTIBODIES 4711 lung adenocarcinoma in 2018. In a meta‑analysis of clinical (Salisbury, UK). The COLO320DM cell line was purchased studies, Li et al (13) showed that aldehyde dehydrogenase 1 from the Japanese Collection of Research Bioresources could be a predictor of response to neoadjuvant chemotherapy Cell Bank (Osaka, Japan). The SW48 cell line was obtained in . Furthermore, in recent years, proteins in from the American Type Culture Collection (Manassas, tumor cells have been comprehensively analyzed. Yu et al (14) VA, USA). These cell lines were classified into three groups identified some predictive marker proteins for sensitivity to according to cetuximab sensitivity: C99 and SW48 in the platinum‑containing drugs in patients with ovarian cancer. cetuximab‑sensitive group (Cmab‑S), C10 and HT55 in the Moreover, Chauvin et al (15) detected predictive marker cetuximab‑partial resistance group (Cmab‑PR), and CACO2 proteins for the efficacy of 5‑fluorouracil (5‑FU) in patients and COLO320DM in the cetuximab‑resistance group with locally advanced rectal cancer. As mentioned above, the (Cmab‑R). Reagents for culture [culture medium, fetal bovine usefulness of proteome analysis of tumor tissues to develop serum (FBS), and supplements] and sample preparation predictive markers for the efficacy of certain small molecule (dithiothreitol, iodoacetamide, and trypsin) were purchased drugs has been demonstrated. On the other hand, although from Wako Pure Chemical Industries (Osaka, Japan). All other L‑lactate dehydrogenase A (LDHA) expression levels in reagents were obtained from commercial sources, and those tumors have been reported to correlate with cetuximab sensi- used to analyze peptides were graded for high‑performance tivity in patients with Ewing's sarcoma (16) and gankyrin has liquid chromatography, liquid chromatography‑tandem mass been reported to contribute to resistance to chemotherapy spectrometry (LC‑MS/MS), or analytical use. Standard containing bevacizumab in CRC (17), comprehensive proteome peptides used for MS analysis were synthesized by Eurofins analysis to identify predictive biomarkers for the efficacy of Genomics Japan (Tokyo, Japan). antibody drugs has not been conducted. Anti‑EGFR monoclonal antibodies (anti‑EGFR mAbs), Cell culture and sample preparation. Six CRC cell lines with including cetuximab and panitumumab, are key drugs in the different sensitivities to cetuximab were used in this study treatment of colorectal cancer (CRC) and are highly effective (Table I) (29). CACO2, COLO320DM, C10, HT55, and C99 cells for some CRC patients. On the other hand, anti‑EGFR mAbs were cultured in a humidified incubator at 37˚C in the presence are very expensive, and are also known to cause serious adverse of 5% CO2, while SW48 cells were cultured in a 37˚C incubator effects such as an infusion reaction or skin rash. Therefore, with no supplemental CO2. Cetuximab‑acquired resistance these drugs should be used only for patients in which an effec- cell lines (SW48R and C99R) were generated upon continuous tive response is expected. Many clinical trials have concluded exposure of SW48 and C99 cell lines to cetuximab according to that variations in the KRAS are a crucial factor affecting the method of Troiani et al (30). Briefly, for a period of 8 months, the clinical efficacy of anti‑EGFR mAbs. Anti‑EGFR mAbs SW48 and C99 cells were continuously exposed to cetuximab have been recommended to be used for wild‑type KRAS CRC to increase the inhibition of 50% of cancer cell growth (IC50), patients, approximately 60% of all CRC patients. However, and the final concentration was 12.8 µg/ml. Cytoplasmic and more than 50% of patients with wild‑type KRAS tumors do not membrane proteins were extracted from 80% confluent cell receive a therapeutic benefit from anti‑EGFR mAbs (18‑22). lines using a Minute Plasma Membrane Protein Isolation kit Even when variations of NRAS, BRAF, and PIK3CA mutations (Invent Biotechnologies, Inc., Plymouth, MN, USA), and the or EGFR overexpression are taken into account, the primary concentrations of the extracted proteins were measured using cause of resistance to anti‑EGFR mAbs in more than half a DC™ Protein assay kit (Bio‑Rad Laboratories, Inc., Hercules, of wild‑type KRAS CRC patients with poor drug responses CA, USA). Cytoplasmic and membrane protein extracts were remains unknown (20,23‑28). As mentioned above, although diluted to 2.0 and 1.0 mg/ml, respectively, and assayed immedi- genome research is widely performed and has many advan- ately or stored at ‑80˚C until assay. tages such as the ease of the procedure, it has become clear that fluctuation in protein expression, which directly affects Mass spectrometry analysis. As a pretreatment for cytoplasmic drug efficacy, cannot be explained with genome information and membrane protein samples, protein samples (45 µl) were alone. Thus, factors affecting drug efficacy other than genetic mixed with 1 mg/ml infliximab (5 µl) and an internal standard factors need to be studied through a comprehensive analysis, (ISTD), and incubated at 37˚C for 30 min with urea (41 mg) such as a proteomic approach. and 40 mg/ml dithiothreitol (7.7 µl) in 8 mol/l urea/0.5 mol/l In this study, we focused on time‑of‑flight (TOF) mass Tris HCl (pH 8.5) to reduce disulfide bonds. Reduced samples spectrometry (MS) which can be used for non‑target protein were alkylated by reaction with 40 mg/ml iodoacetamide in analysis and comprehensive protein analysis, and performed 8 mol/l urea/0.5 mol/l Tris‑HCl (19.2 µl; pH 8.5) for 30 min comprehensive analysis of proteins derived from CRC cell lines at 37˚C. Subsequently, to digest the proteins, cytoplasmic and without genetic mutations affecting sensitivity to anti‑EGFR membrane protein samples (300 µl each, diluted 4‑fold with mAbs, such as KRAS, NRAS, BRAF, and PIK3CA muta- Milli‑Q water) were trypsinized by adding trypsin solution (42 tions, and PTEN overexpression. Finally, we explored some and 22 µl, respectively; 100 µg/ml in 20 mmol/l acetic acid) tumor‑specific proteins that were correlated with sensitivity to and incubating at 37˚C for 12 h. Surfactants in the membrane anti‑EGFR mAbs. protein samples were removed with Detergent OUT™ (Takara Bio, Inc., Shiga, Japan), and trypsinized samples were desalted Materials and methods using a MonoSpin C18 column (GL Sciences, Inc., Tokyo, Japan). Materials. CACO2, C10, HT55, and C99 cell lines were Peptide fragments analysis was performed using liquid purchased from the European Collection of Cell Cultures chromatography‑time‑of‑flight mass spectrometry (LC‑TOF 4712 ONCOLOGY LETTERS 17: 4710-4716, 2019

Table Ⅰ. Cetuximab sensitivities and culture of six CRC cell lines.

Growth inhibition Cell line Cetuximab sensitivity relative to control (%) Culture medium

CACO2 Resistant 0 EMEM + 1% NEAA + 10% FBS COLO320DM Resistant 0 RPMI1640 + 10% FBS C10 Partially resistant 5.9 IMDM + 10% FBS HT55 Partially resistant 21.3 EMEM + 1% NEAA + 20% FBS SW48 Sensitive 69.1 L15 + 10% FBS C99 Sensitive 96.8 IMDM + 10% FBS

CRC, colorectal cancer; NEAA, non‑essential amino acids; FBS, fetal bovine serum.

MS) consisting of an ACQUITY UPLC (Waters, Milford, coefficients ± 0.8 were used to define the peaksassociated with MA, USA) for LC and LCT Premier XE (Waters) for TOF sensitivity to cetuximab. Among the selected peaks, the peaks MS. The LC conditions were as follows: Column, ACQUITY without isotopic peaks were excluded, and the peaks with intensi- UPLC BEH C18 column (2.1 mm x 100 mm, 1.7 µm) (Waters); ties that were significantly correlated with cetuximab sensitivity column temperature, 40˚C; mobile phase, 0.1% formic acid in in the six CRC cell lines, including Cmab‑PR, were finally Milli‑Q water (A) and 0.1% formic acid in acetonitrile (B); extracted. Thereafter, the proteins composing the extracted flow rate, 0.3 ml/min; and gradient program, 5 to 95% B in peaks were determined using MS fit analysis (ProteinProspector; 55 min and 95 to 5% B in 3 min. MS analysis was performed http://prospector.ucsf.edu/prospector/mshome.htm). using an electrospray ionization (ESI) source in positive ionization mode (W mode). Survey scans were acquired in the Statistical analysis. Correlations between the peak intensities range 100 to 2000 m/z. Instrument settings were as follows: and growth inhibitory concentration of cetuximab, an index Capillary voltage, 3000 V; sample cone voltage, 50 V; desol- of cetuximab sensitivity, were evaluated based on Pearson's vation temperature, 350˚C; source temperature, 120˚C; cone correlation coefficient. Differences in LDHB expression levels gas flow, 60 l/h; desolvation gas flow, 700 l/h; and aperture between groups were evaluated by One‑way analysis of vari- 1 voltage, 15 V. ance (ANOVA) and Mann‑Whitney U test. The Tukey‑Kramer Tandem quadrupole MS was used to analyze specific test was used for post hoc analysis. Statistical analysis was peptide fragments. LC was performed with an ACQUITY performed using SPSS software version 24.0 (SPSS, Inc., UPLC system (Waters). An ACQUITY UPLC BEH C18 Chicago, IL, USA). A P‑value less than 0.05 was considered column (2.1 mm x 100 mm, 1.7 µm) (Waters) was used as statistically significant. the LC column. The LC conditions were as follows: Column temperature, 40˚C; mobile phase, 0.1% formic acid in Milli‑Q Results water (A) and 0.1% formic acid in acetonitrile (B); flow rate, 0.5 ml/min; and gradient program, 5 to 35% B in 6 min, 35 to Exploration of predictive marker proteins. PCA was 95% B in 1 min, and 95 to 5% B in 1 min. XevoTQ (Waters) performed on data of the MS peaks obtained from Cmab‑R with ESI turbo spray in positive ionization mode was used, and and Cmab‑S using LC‑TOF MS. A total of 1,599 and 1,012 the ionization parameters were as follows: Capillary voltage, peaks that were specific to cytoplasmic proteins of Cmab‑R 1000 V; desolvation temperature, 500˚C; source temperature, and Cmab‑S, respectively, and 2,370 and 3,478 peaks 150˚C; desolvation gas flow, 1000 l/h; and cone gas flow, that were specific to membrane proteins of Cmab‑R and 70 l/h. The transitions m/z 1176.53>130.85 for LDHB and m/z Cmab‑S, respectively, were detected. Then, we extracted 835.44>175.20 for ISTD were monitored. Sample cone voltage 271 and 197 peaks as monoisotopic ion peaks that were and collision energy were 74 and 80 V for LDHB and 35 and specific to cytoplasmic proteins of Cmab‑R and Cmab‑S, 55 V for ISTD, respectively. respectively, and excluded other peaks as isotopic peaks or noise peaks. Similarly, 533 and 422 peaks were extracted Proteomics analysis. Principal component analysis (PCA) as monoisotopic peaks that were specific to membrane and orthogonal projections for latent structure‑discriminant proteins of Cmab‑R and Cmab‑S, respectively. Finally, 148 analysis (OPLS‑DA) using MarkerLynx XS software (Waters) and 146 peaks from cytoplasmic proteins and 363 and 267 were performed on all peaks obtained from Cmab‑R and peaks from membrane proteins were extracted as candi- Cmab‑S cell lines by LC‑TOF MS analysis with the following date specific peaks to Cmab‑R and Cmab‑S, respectively, parameters: Initial retention time, 0.1 min; final retention time, the intensities of which were significantly correlated with 55.0 min; peak width at 5% height, 3.00 s; peak‑to‑peak base- cetuximab sensitivity in the six CRC cell lines. Candidate line noise, 50.00; mass tolerance, 0.05 Da; intensity threshold, proteins composed of those peaks were identified by MS fit 10 counts; mass window, 0.05 Da; and retention time window, analysis, and LDHB in the cytoplasmic fraction showed the 0.10 min. In OPLS‑DA, the peaks were grouped into Cmab‑R highest MOWSE score, an indicator of the probability of and Cmab‑S, and cut‑off values for Pearson's correlation protein identification (Table II). NAGAMINE et al: LDHB AS A PREDICTIVE MARKER FOR SENSITIVITY TO ANTI-EGFR MONOCLONAL ANTIBODIES 4713

Table II. Candidate marker proteins for cetuximab sensitivity.

MOWSE No. score % Coverage Protein name Accession no. Fraction Marker

1 7198 14.4 L‑lactate dehydrogenase B chain P07195 Cytoplasm Cmab‑R 2 911 10.4 Uncharacterized protein C18orf63 Q68DL7 Membrane Cmab‑S 3 573 7.1 /threonine‑protein kinase TAO1 Q7L7X3 Membrane Cmab‑S 4 303 4 Protein phosphatase Slingshot homolog 1 Q8WYL5 Membrane Cmab‑S 5 273 4.9 Coiled‑coil domain‑containing protein 110 Q8TBZ0 Cytoplasm Cmab‑S 6 242 5.4 Pre‑mRNA‑splicing factor ATP‑dependent O60231 Membrane Cmab‑S RNA helicase DHX16 7 240 4.6 Exocyst complex component 6B Q9Y2D4 Membrane Cmab‑S 8 237 7.9 Zinc finger protein 771 Q7L3S4 Membrane Cmab‑R 9 224 13.5 Protein LIAT1 Q6ZQX7 Membrane Cmab‑S 10 223 8.7 Kelch‑like protein 41 O60662 Membrane Cmab‑S

MOWSE, molecular weight search; Cmab‑S, cetuximab‑sensitive group; Cmab‑R, cetuximab‑resistance group.

Evaluation of the correlation between peak intensity of an In 2012, LDHB expression was found to be correlated with LDHB‑specific peptide and cetuximab sensitivity. From the cell growth in lung cancer with KRAS mutations (41), and its final candidate peaks, six (m/z 720.40, 957.62, 754.38, 1011.57, usefulness as a prognostic factor for triple‑negative breast 959.56, and 1176.60) were extracted as peptide peaks derived cancer, osteosarcoma and NSCLC was also reported by from LDHB by MS fit analysis, and an LDHB‑specific peptide, Li et al (35), McCleland et al (38), and Koh et al (42). Moreover, SADTLWDIQK m/z 1176.60, was analyzed by LC‑MS/MS the effect of LDHB on the efficacy of some anticancer drugs (Fig. 1). As a result, the peak intensity of SADTLWDIQK has been investigated, and LDHB expression levels are was significantly higher in Cmab‑R than in Cmab‑S (P<0.05, reported to be correlated with the pathological complete One‑way ANOVA) (Fig. 2). Only C10, but not C99, was response ratio, disease‑free survival, and poor overall survival significantly higher in Cmab‑PR than in Cmab‑S (P<0.05, in patients treated with small molecule anticancer drugs, such One‑way ANOVA) (Fig. 2). In addition, the data obtained by as anthracycline and taxane (11,43). LC‑MS/MS were consistent with data obtained by analysis The activation of LDH has been reported to enhance the using enzyme‑linked immunosorbent assay (ELISA) (data not Warburg effect (11,44,45). The Warburg effect causes the acti- shown). vation of intracellular stress signaling pathways by increasing glucose consumption and inducing hypoglycemia, and its acti- Relationship between acquired cetuximab resistance and vation is also known to lead to the development of resistance LDHB expression. We compared the expression levels of LDHB against anticancer drugs, such as 5‑FU, carboplatin, and topoi- in the SW48R and C99R cell lines, which acquired resistance somerase inhibitors (46‑48). Furthermore, enhanced heat shock to cetuximab, and in the original cell lines. LDHB expression protein 90 activation, a molecular chaperone evoked during levels were significantly upregulated in the CRC cell lines that stress reactions, by the Warburg effect has also been reported to developed cetuximab resistance when compared to the original be involved in the development of resistance to anticancer drugs cell lines (P<0.05, Mann‑Whitney U test) (Fig. 3). In addition, by activating client proteins, such as PI3K/Akt (49‑51). the data obtained using LC‑MS/MS were consistent with data Unlike many previous studies of the effects of LDHB obtained by analysis using ELISA (data not shown). expression on prognosis or response to chemotherapy, which focused on LDHB alone, we demonstrated that the level of Discussion LDHB expression correlated with sensitivity to anti‑EGFR mAbs by comprehensive analysis of proteins derived from We showed that the expression level of LDHB may be a CRC cell lines. Furthermore, LDHB expression levels were predictive biomarker for sensitivity to anti‑EGFR mAbs by significantly upregulated with the acquisition of resistance to proteome analysis for the first time. Furthermore, our find- cetuximab in Cmab‑S CRC cell lines. These results indicated ings suggested that LDHB may also be involved in acquired that high LDHB expression was particularly important for resistance to anti‑EGFR mAbs. the acquisition of drug resistance among many key factors LDH is a key glycolytic enzyme catalyzing the conversion involved in the development of drug resistance. In contrast, the of pyruvate acid to lactic acid, and is composed of two major LDHB expression level was low in the partially resistant HT55 subunits, LDHA (muscle type, M subunit) and LDHB (heart cell line, and these data indicated that cetuximab sensitivity type, H subunit). LDHA is reported to be involved in cell could not be evaluated with LDHB expression level alone. proliferation and metabolism in most cancers (31‑34), while Although the cause of partial resistance to cetuximab in HT55 LDHB expression varies greatly among cancer types (35‑40). has not been clarified, various factors have been reported to 4714 ONCOLOGY LETTERS 17: 4710-4716, 2019

Figure 1. Candidate fragment ions derived from LDHB by MS fit analysis. LDHB chain sequence and six candidate fragment ions (m/z 720.40, 957.62, 754.38, 1011.57, 959.56 and 1176.60) derived from LDHB determined by conducting MS fit analysis using LC‑TOF MS. No trypsin digestion mistakes in the candidate ions were detected.

Figure 2. Expression levels of an LDHB‑specific sequence using LC‑MS/MS in cytoplasmic proteins derived from six CRC cell lines. (A) LC‑MS/MS chromatogram of the LDHB‑specific sequence (SADTLWDIQK) in six CRC cell lines. (B) Peak area ratios of the LDHB‑specific sequence (SADTLWDIQK) and the internal standard in six CRC cell lines. Data are presented as the mean ± standard deviation of triplicate experiments (One‑way ANOVA, *P<0.05).

caused by many factors in addition to the increase of LDHB. Further detailed investigations are needed to examine drug resistance in this cell line. One limitation of our study is that we could not clarify whether the drug resistance caused the increase in LDHB levels or whether increased levels of LDHB led to drug resis- tance. As mentioned above, LDHB is known to be an important factor related to the Warburg effect, and LDHB expression in tumor cells is reported to be increased to facilitate survival in hypoxic and low‑energy conditions associated with insuf- Figure 3. Changes in expression level of LDHB after acquisition of cetux- ficient angiogenesis. Since our results are based on an in vitro imab resistance. Changes in LDHB expression levels in cytoplasmic proteins derived from cetuximab‑sensitive cell lines (SW48 and C99) and cell lines study, the survival environment of cancer cells is considered that acquired cetuximab resistance (SW48R and C99R). Peak area ratios to be unchanged at least with respect to oxygen concentration between the LDHB‑specific sequence (SADTLWDIQK) and the internal and nutritional condition. In addition, LDHB protein level was standard in four CRC cell lines. Data are presented as the mean ± standard * low in the partially resistant HT55 cell line. According to these deviation of triplicate experiments (Mann‑Whitney U test, P<0.05). data, resistance to cetuximab was considered to be developed following changes associated with increased LDHB expression levels but LDHB protein level did not increase with the acqui- be associated with resistance to cancer chemotherapy. Since sition of resistance to cetuximab. The report by Sun et al (11) primary resistance to drugs is known to be caused by so many showed that LDH B deletion sensitized ora l squa mous cell ca rci- factors, primary resistance to cetuximab in HT55 could be noma cell lines to taxane, whereas the introduction of LDHB NAGAMINE et al: LDHB AS A PREDICTIVE MARKER FOR SENSITIVITY TO ANTI-EGFR MONOCLONAL ANTIBODIES 4715 decreased sensitivity to taxane. Furthermore, Lu et al (52) 2. Li B, Li C, Guo M, Shang S, Li X, Xie P, Sun X, Yu J and Wang L: Predictive value of LDH kinetics in bevacizumab treatment also reported that cetuximab showed antitumor efficacy due and survival of patients with advanced NSCLC. Onco Targets to downregulation of the α subunit of HIF‑1 (HIF‑1α). This Ther 11: 6287‑6294, 2018. in turn regulated the expression of LDHA and inhibition of 3. Gregorc V, Novello S, Lazzari C, Barni S, Aieta M, Mencoboni M, Grossi F, De Pas T, de Marinis F, Bearz A, et al: Predictive glycolysis in cetuximab‑sensitive head and neck squamous value of a proteomic signature in patients with non‑small‑cell cell carcinoma cells in a HIF‑1α downregulation‑dependent lung cancer treated with second‑line erlotinib or chemotherapy manner; these reports support our hypothesis. However, we (PROSE): A biomarker‑stratified, randomised phase 3 trial. Lancet Oncol 15: 713‑721, 2014. could not prove our hypothesis with our data, and so a more 4. Lazzari C, Spreafico A, Bachi A, Roder H, Floriani I, Garavaglia D, detailed study of the role of LDHB in resistance to cetuximab Cattaneo A, Grigorieva J, Viganò MG, Sorlini C, et al: Changes in is needed. 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